Project Summary/Abstract: Sickle cell disease (SCD) is the most common genetic disorder worldwide. Bone loss is a common complication in humans with SCD that predispose them to increased fractures, vertebral collapse, and bone pain. Eighty percent of adults with SCD have low bone mineral density that is independent of usual risk factors. This suggests the cause of bone loss in SCD differs from the general population. However, the mechanism(s) of bone loss in human SCD has not been fully investigated, and there are no targeted therapies to prevent or treat compromised bone health in this population. Recent studies showed gut microbiota-derived signals regulate neutrophil aging in blood that mediates the inflammation-related liver and spleen damage in SCD mice. However, the connection between the gut microbiota and sickle cell bone loss is unknown. Our preliminary data showed significantly reduced bone mass in SCD mice, a striking change in the composition of the gut microbiota, and an overall increase in gut bacteria load; interestingly, depletion of gut microbiota with antibiotics significantly improved bone mass in SCD mice and bone phenotype is transmissible between Ctrl and SCD upon co-housing. Compositional changes of gut microbiota included a marked increase in potentially pathogenic Peptostreptococcaceae family bacteria. Several species from this family possess high nucleotide- binding oligomerization domain-containing protein 1 (NOD1)-stimulatory activity that is known to regulate bone loss. Increased Peptostreptococcaceae family bacteria was accompanied by increased NOD1 ligand in serum and Nod1 mRNA in bone of SCD mice, providing a link between these two observations. Therefore, Aim 1 proposes a mechanistic study of the pathogenic gut bacteria induced bone loss in SCD mice. In addition to increases in potentially pathogenic bacteria, we observed a marked decrease in the relative abundance of potentially beneficial Coriobacteriaceae family bacteria, several species of which can convert food polyphenols to S-equol, a bone protective compound in humans and rodents. Reduced S-equol in serum of SCD mice provided compelling support for this initial finding. S-equol is an estrogen receptor agonist, decreased S-equol can decrease serum IGF-1, and serum and bone IGF-1 were decreased in SCD mice (our published data). Thus, Aim 2 proposes a mechanistic study of decreased protective gut bacteria leading to bone loss in SCD mice. Our in vitro data demonstrated an accumulation of aged neutrophils in bone marrow (BM) and a direct interaction between these neutrophils and osteoblasts (OBs)/osteoclasts (OCs), which was associated with impaired OB function/enhanced OC formation. Aim 3 proposes an in vitro mechanistic study of how BM neutrophils from SCD mice affect OB and OC functions. Our goals are to investigate how the gut microbiota contributes to bone loss in SCD and to identify pathogenic and protective bacterial strains that could be targeted for future therapies. Our work will provide valuable preclinical data to determine if modulation of the gut microbiome can act as potential therapies to prevent and treat bone loss in human SCD.